Preliminary study on the detection of hydrogen ion flux in breast cancer tissue using noninvasive microtest technology

doi:10.21203/rs.3.rs-2553939/v1

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Preliminary study on the detection of hydrogen ion flux in breast cancer tissue using noninvasive microtest technology

https://doi.org/10.21203/rs.3.rs-2553939/v1

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Objective

To detect the extracellular hydrogen ion (H+) flux of breast cancer tissue explants using noninvasive microtest technology (NMT) and assess how this flux correlates with the molecular subtype. Further more, we preliminarily explored the possibility of applying NMT as a treatment prescreening tool for predicting how docetaxel will affect tissue responses.

Methods

This study enrolled 30 breast cancer patients who underwent surgery in the Department of Breast Surgery at Beijing Hospital, Beijing, China. Tumor samples and the corresponding normal samples were excised from surgical specimens with a size of approximately 0.5 cm³. The NMT system was used to detect the H⁺ flux of tumor samples and corresponding normal samples. Then, the NMT system was used to assess the changes in H + flux after the tumor samples were treated with 10 mg/L docetaxel.

Results

All the samples produced stable H + flux that could be measured in real time. In 26 cases, we found that tumor samples exhibited stable and robust efflux of H+, whereas in the corresponding normal samples, we measured significant differences with H⁺ influx or mild efflux (cancer samples: 0.336 ± 0.307 pmol/cm^− 2/sec^− 1 vs. normal samples: -0.067 ± 0.131, p < 0.001). Among the 26 tumor samples that showed efflux of H⁺, there were 4 luminal A type, 11 luminal B type, 6 HER2-positive type and 5 triple-negative type. Triple-negative tumors had a higher rate of H + efflux compared with luminal A, luminal B and HER2 positive tumors (p = 0.016, p = 0.018, p = 0.008). Among the 4 tumor samples that were treated with docetaxel, 3 samples showed inhibition of H + efflux by 50.5%, 28.8% and 8.3%, while efflux increased by 30.0% in 1 sample.

Conclusion

NMT can detect the H + flux of breast cancer tissue in real time. The H + flux of tumor samples was significantly different from that of normal samples. After treatment with docetaxel, the H + flux of tumor samples showed definite changes.

Noninvasive microtest technology

H+ flux

breast cancer

molecular typing

docetaxel

Previous studies have shown that tumor cells can produce a large amount of lactate and carbonic acid through the Warburg effect and overexpression of carbonic anhydrase [1]. In addition, H⁺-related transporters such as Na+/H + exchange proteins, Na+/K+-ATPase and monocarboxylic acid transporters are upregulated in tumor cells. These changes promote the efflux of H + and ultimately support the development of a weak alkaline environment in the cells and an acidic microenvironment outside the cells[2, 3]. The weakly alkaline intracellular environment supports the proliferation and glycolysis of tumor cells, while the extracellular acidic microenvironment is conducive to the migration and invasion of tumor cells [4, 5]. At the cellular level, the detection of ions and molecules can be accomplished by techniques such as patch clamp and laser confocal [6, 7, 8]. Considering that the microenvironment is formed via the joint action of a variety of cells, stromal components and cytokines [9], the ion conductance of a single cell may not reflect the overall situation of the tumor tissue environment. Therefore, a tissue-level technique is needed to measure the transport of ions and molecules.

Noninvasive microtest technology (NMT) is a highly sensitive technique that can detect the concentration gradient of ions or molecules outside the sample in a noncontact manner. It can be used in organelles, cells, tissues, organs and individual local sites[11–14]. The technique can be used to track and measure a wide array of analytes, including ions and small molecules, without any chemical staining, such as fluorescence and radioisotope labeling. This technology can measure ions or molecules related to the physiological activities of tissues automatically by computer and precision motion control systems without touching the tissues, thus avoiding the possibility of data artifacts caused by cell injury [15]. Due to its ability to yield real-time, in situ, quantitative and relatively long-term continuous measurements, NMT has become increasingly emphasized as an indispensable technique for studying the physiological activities of living systems.

Breast cancer is the most common malignant tumor in the world. Although the development of comprehensive treatments has significantly improved the prognosis of breast cancer patients, some patients still experience recurrence and metastasis after initial treatment. Clinically, breast cancers are classified into different molecular types according to pathological tests. Different molecular types of tumors show different biological characteristics such as proliferation and metastatic potential. However, whether different subtypes have different physiological characteristics in the tumor microenvironment is poorly understood due to the lack of effective detection and evaluation methods that address the biophysics of the cancer microenvironment.

In addition, adjuvant neoadjuvant therapies are important means of breast cancer treatment, and better methods to determine how to apply effective drugs in a timely and accurate manner are needed [10]. Previous in vitro drug sensitivity tests included the tetrazolium blue colorimetric method, the cytotoxicity differential staining method, the thymine nucleoside incorporation method, and the ATP bioluminescence method[16]. Most of these tests are performed at the cellular level and limited by sensitivity, detection time, detection efficiency and other factors. To carry out targeted therapy for patients as early as possible, a detection method that can rapidly obtain accurate drug susceptibility information is urgently needed in clinical practice.

In this study, we aimed to use the NMT technique to detect H + flux in breast cancer tissue and to use it as a tool for drug susceptibility assays.

1. Patients and ethics

Thirty breast cancer patients who underwent surgery in Beijing Hospital from May 2022 to August 2022 were consecutively selected. The inclusion criteria included the following: (1) invasive breast cancer confirmed by core needle biopsy, (2) tumor size ≥ 1 cm, (3) mastectomy, And (4) agreement to provide specimens and signed informed consent. The exclusion criteria included the following: (1) use of preoperative neoadjuvant therapy, (2) previous breast-conserving surgery, and (3) refusal to sign the consent form and provide specimens.

The study protocol was approved by the Ethics Committee of Beijing Hospital on the basis of the Declaration of Helsinki (IRB Number in Ethical approval: 2022BJYYEC-130-02), and written informed consent was obtained from the patients.

2. Tissue sampling and processing

Tumor samples and corresponding normal samples were excised from surgical specimens, with a length of approximately 0.5 cm×0.5 cm×0.5 cm. The tumor sample was excised from the tumor center, and the normal tissue was excised from the normal gland more than 2 cm away from the tumor. The samples were placed in RPMI-1640 medium containing 10% calf serum and double antibody (penicillin + streptomycin) and stored in a 4°C ice box. The surface of each tissue near the center of the tumor was defined as the detection surface (Figure 1A). An equilateral triangle was sketched on the measurement surface, and the three fixed points of the triangle were used as detection site (Figure 1B).

3. Detection of H+ flux in tumor samples and normal samples

(1). An NMT workstation (Xuyue (Beijing) Technology Co., Ltd.) was used to detect the H+ flux in real time. The workstation included imFluxes V2.0 software (YoungerUSA LLC, Amherst, MA 01002, USA), integrated voltage signal acquisition, 3D motion control, and image acquisition.

(2). Preparation of H+ sensor: Microsensors (Φ5 ± 1 μm, XY-CGQ-01, YoungerUSA) were drawn, silanized and filled with electrolyte (15 mM NaCl + 40 mM KH2PO4, pH 7.0) to a length of 1 cm from the tip of the microsensor. A 40-50 µm column of selective liquid ion exchangers (H+ LIX, XY-SJ-H, YoungerUSA) was packed in front of the microsensor. Insert the Ag/AgCl glass microflux sensor holder (NMT-HC-32) from the tail of the H+ sensor to make full contact with the electrolyte. An Ag/AgCl ultralow permeability solid reference electrode (NMT-HC-86) was used as the reference electrode.

(3). Calibration of H+ sensor: The H+ sensor was calibrated at three different H+ concentrations: pH 6.5, pH 7.2 and pH 7.5. Then, we detected the H+ flux by the sensor with the Nernst slope in the range of 58 ± 5 mV/decade. After each test, the same sensor was recalibrated according to the same procedure and standard. The data will be discarded if the calibration fails after the test,

(4). Sampling rule selection: According to the formula J = −D0·(dc/dx), dx is the distance that the microsensor moves from a point close to the sample to another point vertically away from the sample surface at a certain frequency. dx is usually 5 - 35 μm, and 30 µm was used in our study.

(5). Detection of H+ flux: All tests were completed within 2 hours after the specimen was isolated. First, filter paper strips and resin blocks were used to attach tumor or normal samples to the bottom of the dish, exposing the top surface of the samples. Second, the test solution (0.1 mM CaCl2, 0.5 mM KCl, 137 mM NaCl, 0.1 mM NaHCO3, 0.1 mM KH2PO4, 0.1 mM Na2HPO4, 5.6 mM glucose, pH 7.2) was added to the dish until the samples were submerged. After ten minutes, the test solution was discarded, and 8 ml of new test solution was added. Third, the H+ flux rate sensor was placed at a distance of approximately 50 µm from the detection site on the sample surface under the microscope. Each site will be detected for 3 minutes. The H+ flux rate was directly detected via Imfluxes V2.0 software, and the H+ flux unit was mol∙cm-2∙s-1. Positive values represent efflux, and negative values represent influx. The composition of the NMT system is shown in Figure 2.

4. Treatment with docetaxel

We randomly chose 4 tumor samples from those with H+ efflux. First, the sample was detected for 3 minutes to obtain stable H+ flux. Then, 10 mg/L docetaxel(Aventis Pharma S.A. France) was added to the petri dish. After the the signal was stable, H+ flux was recorded for 3 minutes.

5. Pathological examination

After the detection of H+ flux was completed, the tissue samples were labeled with nanocarbon on the opposite side of the test surface and fixed with 10% neutral formalin solution. All pathological examinations were performed in the Pathology Department of Beijing Hospital. The inspection included tumor histological type, histological grade, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth receptor-2 (HER-2) status, and Ki-67 index. According to the 2020 CAP clinical guidelines, ER/PR positivity was defined as tumor cell expression of 1% - 100% [17]. According to the 2018 ASCO/CAP guidelines. HER-2 positivity was defined as IHC 3+ or IHC 2+ with ISH positivity[18]. Four molecular subtypes (luminal A, luminal B, HER-2 positive and triple-negative breast cancer), were classified according to ER, PR, HER-2 and Ki67 expression [19].

6. Statistical methods

SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was used for analysis. H+ flux was described according to the mean±standard deviation. The differences in H+ flux between tumor samples and normal samples were compared by independent sample t tests. One-way ANOVA was used to compare the differences among molecular subtypes. For all tests, differences were considered to be statistically significant at p < 0.05.

Patients and tumors

We enrolled 30 breast cancer patients between May 2022 and August 2022. All patients were female with an mean age of 56.1 ± 11.9 years. 27 cases were invasive ductal carcinoma, 2 cases were invasive lobular carcinoma, and 1 case was adenoid cystic carcinoma. After NMT detection, all the tumor samples were pathologically confirmed to have tumor cells in detection surfaces, and the detection surfaces of normal samples were free of tumor cell. Figure 3 shows the histopathological images of the detection surface of the cancer sample and the corresponding normal sample.

NMT can detect stable H+ flux in all samples

Using NMT, we recorded the rate and direction of H+ flux in real time. The detected value was the result obtained by subtracting the influx rate of H+ from the efflux rate of H+. When the detected value is positive, it indicates the net efflux of H+. When the detected value is negative, it means the net influx of H+. Taking case 6 as an example, we recorded stable H+ flux in real time (Figure 4).

Figure 5 shows the mean rate of H+ flux in tumor samples and normal samples across all 30 cases. In 26 cases (86.7%), the H+ flux in tumor samples always showed efflux (0.385 ± 0.200 mol∙cm-2∙s-1), while mild efflux or influx was observed in normal samples (-0.019 ± 0.062 mol∙cm-2∙s-1). The differences were statistically significant (p < 0.001). In 4 cases, mild H+ influx was observed in both tumor samples and normal samples (-0.217 ± 0.216 mol∙cm-2∙s-1 vs. -0.154±0.133 mol∙cm-2∙s-1, Figure 6B).

H+ flux was correlated with molecular subtype

We analyzed the molecular subtypes of 26 tumor samples with H+ efflux (Table 1). 4 cases were luminal A type with H+ flux rates of 0.046 ± 0.043 mol∙cm-2∙s-1. 11 cases were luminal B type with H+ flux rates of 0.297 ± 0.233 mol∙cm-2∙s-1. 6 cases were HER2-positive with H+ flux rates of 0.316 ± 0.105 mol∙cm-2∙s-1. 5 cases were triple-negative breast cancer with H+ flux rates of 0.847 ± 0.274 mol∙cm-2∙s-1. Compared with luminal A, luminal B and HER2-positive breast cancer, triple-negative breast cancer had higher H+ efflux rates (p = 0.016, p = 0.018, p = 0.008).

H+ flux changed after docetaxel treatment

Real-time changes in H⁺ flux in 4 tumor samples after docetaxel treatment were recorded. Three samples showed decreases in mean rate of H⁺ flux (50.5%, 28.8% and 8.3% decreases) and one sample showed a 30% increase (Table 2).

NMT is carried out in a liquid that simulates the real living environment of biological samples without introducing any exogenous substances, which ensures the preservation of the biological activity and physiological state of the samples to the greatest extent. The greatest difference from other invasive measurement techniques is that the NMT test process is automatically controlled by the computer to control the movement of the microelectrode, which is very close to the sample surface without touching the sample. NMT can simultaneously determine the absolute concentration, flux rate and flux direction of ions/molecules on the surface of samples[20]. Because the microelectrode does not need to contact the sample during the test, the application range of NMT is very wide, from animal and plant organs, tissues, cell aggregates, and single cells to enriched organelles. Thus far, NMT has been applied to measure the flux of ions and molecules such as Ca2+, H+, O2, H2O, K+, Mg2+, Na + and NH4 + in animal and plant tissues and cells [11–14]. In the animal field, previous studies have mostly been performed at the cellular level, and there is still a lack of sufficient research on detection at the tissue level.

The flux rate and flux direction information for ions/molecules obtained by NMT not only reflects the dynamic process of biological and physiological activities but also reflects general features of the assessed tissues/cells. For example, the flux rate of ions/molecules in cells is the result of the joint action of several ion channels and ionophore or molecular transport processes on the cell membrane. The tissue ion molecule flux rate reflects the result of the joint action of several cells in the tissue and is not affected by factors such as the mode of ion molecule transport and whether the transport process is electrically neutral. Thus, the ionic/molecular flux information obtained by NMT provides a very comprehensive and direct means to characterize biological functions and physiological mechanisms.

In our study, we detected stable H + flux in tumor samples and normal samples using NMT. Among most cases, the tumor samples showed H + efflux, while the corresponding normal samples showed mild H + influx (P < 0.001). The obvious efflux of H + from cancer tissue is also consistent with the Valsalva effect and the formation of an acidic microenvironment in cancer tissue based on previous theories and studies [2, 3]. Previous studies have shown that the acidic microenvironment outside the tumor tissue creates favorable conditions for cancer cell migration and invasion[4]. However, the relationship between the intensity of H + efflux and the invasiveness of cancer has not been studied. Through this experiment, we believe that the use of NMT can solve the problem of quantitative detection of H + efflux at the histological level. The results showed that higher H + flux rates were detected in breast cancer tumor samples with unfavorable biological characteristics, such as HER2 positivity and triple negativity.

Previous studies have shown that the acidic microenvironment of tumors can isolate chemotherapeutic drugs in the acidic zone chamber and upregulate the activity of glycoproteins, making drugs unable to penetrate the lipid layer of the cell membrane into cells and ultimately leading to drug resistance[21, 22, 23]. Song Jin et al. performed a preliminary study on H + and drug resistance in breast cancer. The results showed that there was a relationship between tumor H + efflux and drug resistance to doxorubicin[24]. However, H + flux was detected in cancer cells but may not reflect the changes in H + flux in living tissue under the action of chemotherapeutic drugs. In our study, the efflux of H + decreased in 3 breast tumor samples and increased in 1 sample. In addition, H + flux can remain relatively stable during the detection process, which indicates that cancer tissue can maintain a relatively stable biological activity for a period of time after isolation. The changes in H + flux may reflect the sensitivity of cancer samples to chemotherapy drugs. Our approach allowed us to measure the changes in H + flux in tumor tissue after treatment with chemotherapeutic drugs. Based on these results, we can speculate that NMT can be used as a rapid detection technique for drug susceptibility.

Our study has several limitations that need to be addressed. First, although most of the cancer samples showed H + efflux, 4 cases showed H + influx, which lacked an adequate explanation. Second, three of the four specimens showed a decrease in H + efflux after docetaxel treatment, but whether this can be used as an indicator of drug sensitivity needs to be examined in more clinical trials.

In summary, we applied NMT to detect H + flux in breast cancer tissue for the first time, and we detected steady H + flux in real time. We found that luminal B and triple-negative breast cancer had a higher H + efflux rate than luminal A breast cancer. After treatment with docetaxel, the H + flux of cancer tissue showed definite changes.

Acknowledgements

Not applicable.

Authors contributions

All authors contributed to the study conception and design. YX, LX, LY and HB were involved in subject recruitment and surgery. YB, ZZ and YB were involved in detection of H+ flux. LY, BH and Porterfield DM had a big help in collecting clinical data and analysing the data. YX and LX wrote the manuscript. All authors have read and approved the manuscript.

Funding

This study was supported by Beijing Hospital Clinical research 121 Project, Project No.: BJ-2019-191.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The study protocol was established according to the ethical guidelines of the Helsinki Declaration and approved by the Ethics Committee of Beijing Hospital (IRB Number in Ethical approval: 2022BJYYEC-130-02), and written informed consent was obtained from the patients..

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1. H+ flux in different molecular types of breast cancer

Molecular typing	N	H+ flux	p value
luminal A	4	0.054 ± 0.057	0.016
luminal B	10	0.323 ± 0.242	0.018
HER2-positive	10	0.400 ± 0.396	0.008
triple-negative	2	0.625 ± 0.017

Bold figure note: this variable is statistically significant.

Table 2. Changes of H+ flux after docetaxel treatment

	case1	case2	case3	case4
H+ flux before docetaxel treatment	0.4884	0.1345	0.4489	0.2722
H+ flux after docetaxel treatment	0.2418	0.1233	0.5836	0.1937

No competing interests reported.

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Preliminary study on the detection of hydrogen ion flux in breast cancer tissue using noninvasive microtest technology

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